Oxygen homeostasis is essential for life and health, including pulmonary and cardiovascular health. Chronic hypoxia is associated with a myriad of pathologies including pulmonary related diseases such as COPD, lung cancer, pulmonary hypertension, fibrosis and inflammation. Research on oxygen sensing pathways has revealed important mediators of oxygen homeostasis, however, detailed knowledge of the molecular mechanisms of oxygen sensing remain uncovered. Fuller understanding of the cellular oxygen sensing pathways may lead to identification of novel therapeutic targets for treatment of pulmonary and cardiovascular disease. At the cellular level, hypoxia activates the Hypoxia Inducible transcription Factors (HIFs). HIFs bind to hypoxia-response elements in the promoter/enhance regions of a large number of target genes resulting in activation of a genetic program that includes upregulation of glycolysis, angiogenesis, and erythropoiesis. Under normoxic conditions, the HIF protein subunit is hydroxylated by Prolyl Hydroxylase Domain protein 2 (PHD2), targeting it for ubiquitination by the von Hippel-Lindau (VHL) ubiquitin ligase, and proteasomal degradation. The activity of PHD2 is inhibited under hypoxic conditions, allowing the accumulation of HIF protein and the subsequent binding to HIF subunit to induce the transcriptional response. The underlying mechanism by which oxygen levels diminish PHD2 activity is not fully understood. We have previously demonstrated that hypoxia increases the generation of ROS from mitochondrial complex III resulting in inhibition of HIF protein hydroxylation and subsequent HIF protein stabilization. In this proposal we will utilize a proteomic approach to test whether hypoxia-induced mitochondrial ROS lead to inhibition of PHD2 activity by binding of unidentified proteins, post-translational modification of PHD2, and/or oxidizing of cysteine residues within PHD2. Currently, there have been no successful drugs in the clinic targeting inhibition of HIFs. This exploratory grant will provide detailed understanding of how PHD2 is regulated during hypoxia leading to rationale molecular basis for therapeutic targeting of HIFs.